A. Goto

Long-term infusion of kallikrein attenuates renal injury in Dahl salt-sensitive rats.

We investigated whether long-term infusion of kallikrein would attenuate renal injury in salt-induced hypertension in Dahl salt-sensitive rats. A subdepressor dose of purified rat urinary kallikrein (700 ng/d IV) was infused by osmotic minipump for 4 weeks in male Dahl salt-sensitive rats fed a high salt (2% NaCl) diet. This dose did not affect the time-dependent elevation of blood pressure; however, urinary protein excretion was significantly decreased, and glomerular filtration rate was increased. These beneficial effects were reflected morphologically by an attenuation of glomerulosclerotic lesions and tubular injury seen in the hypertensive Dahl salt-sensitive rats. Kallikrein infusion increased urinary excretion of bradykinin and stimulated excretion of cyclic GMP, suggesting that the kallikrein-kinin-prostaglandin and nitric oxide axes were enhanced by rat urinary kallikrein infusion. The alterations induced by kallikrein infusion were potentiated by the concomitant administration of the angiotensin-converting enzyme inhibitor alacepril. These studies indicated that long-term replacement with rat tissue kallikrein attenuates renal injury in hypertensive Dahl salt-sensitive rats.

Presence of digitalis-like factor in mammalian plasma

We attempted to purify a digitalis-like factor from volume expanded dog plasma using an inhibitory effect on the binding of [3H]ouabain to intact human erythrocytes to monitor digitalis-like activity. A highly polar [3H] ouabain displacing compound was purified to a high degree using a combination of chromatographic procedures including reverse phase and gel filtration high performance liquid chromatography. This compound, a reversible inhibitor of [3H]ouabain binding, closely resembles ouabain in its polarity and significantly increases during saline infusion. Its molecular weight was estimated to be 343Da. Moreover, similar compound was consistently detected in other mammalian plasma.

Purification and characterization of human urine-derived digitalis-like factor

We were able to purify a digitalis-like factor to apparent homogeneity from human urine based on the inhibitory effect on [3H]-ouabain binding to intact human erythrocytes. This ouabain displacing compound closely resembles ouabain in its polarity, molecular weight, non-peptidic nature and mode of action except for its UV absorbance spectrum. This compound sharing many biological activities of ouabain may be the endogenous ligand for the Na+, K+-ATPase and serve as a specific regulator of the sodium pump.

Urinary sodium pump inhibitor raises cytosolic free calcium concentration in rat aorta.

We were able to purify two distinct sodium pump inhibitors to homogeneity from human urine based on [3H]ouabain-displacing activity from intact human erythrocytes. The polar and less polar compounds were eluted off the C18 reverse-phase column with 18% and 31% acetonitrile, respectively. The polar compound cross-reacted very weakly with specific antidigoxin antibody and lacked a characteristic ultraviolet absorption peak between 190 and 300 nm. The less polar compound showed a prominent digoxinlike immunoreactivity and had an ultraviolet spectrum similar to that of digoxin. We examined the effects of these compounds on cytosolic free calcium concentration in cultured rat vascular smooth muscle cells (AlO cells) using the fluorescent calcium chelator fura-2. Only the polar ouabain-displacing compound caused a significant increase, from 108±7 to 162±8 nM (n = 6, p < 0.01), in cytosolic free calcium concentration in AlO cells. The rise in cytosolic free calcium concentration induced by the polar ouabaindisplacing compound tended to be slower in onset and more sustained than that induced by arginine vasopressin. In contrast, ouabain and bufalin had no appreciable effects on cytosolic free calcium concentration in AlO cells. These results suggest that the polar ouabain-displacing compound we isolated from human urine may possess a vasoactive property and may play an important role in the modulation of vascular tone.

Existence of a polar digitalis-like factor in mammalian hypothalamus.

We were able to partially purify a polar digitalis-like factor from rat and bovine hypothalami based on the capacity to inhibit [3H]ouabain binding to intact human erythrocytes. This factor was characterized in reference to the digitalis-like factor that we have isolated and reported on. Hypothalamic factor shared digitalis-like activities and physicochemical properties with the one derived from human urine and mammalian plasma. These findings strongly suggest that a polar digitalis-like factor identical to the circulatory factor does exist in mammalian hypothalamus.

Does Digoxin-Like Immunoreactivity Really Represent the Natriuretic Hormone?

Atsuo Goto, MD, Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo,7-3-1 Hongo, Bunkyoku,Tokyo 113 (Japan) Dear Sir, The possibility that endogenous inhibitors of the sodium pump exist and bind to the cardiac glycoside receptor on Na+, K+-ATPase has been a source of much interest. The wide distribution of the high affinity binding site for the cardiac glycoside in normal tissues strongly support this speculation. There is increasing recognition that such endogenous digitalis-like factors may act as ‘natriuretic hormones’ by modulating sodium pump activity in response to extracellular volume expansion and play roles in the pathogenesis of hypertension or chronic renal failure [1, 2]. Many investigators have measured digoxin-like immunoreactivity to identify the putative digitalis-like factors on the assumption that biological substances which bind to the same receptors as drugs may compete with antibodies specific for the drugs. However, it has become evident that the use of antidigoxin antiserum in radioim-munoassay (RIA) systems to detect digitalis-like factors may give some erroneous findings and difficulties in the interpretation of results [3]. Many steroids give false-positive results for digoxin, as do many lipids and bile acids. Recent findings indicate the dissociation of digoxin-like immunoreactivity from digitalis-like biological activity [3–5]. We have recently purified to homogeneity two distinct digitalis-like factors from human urine based on [3H]oua-bain displacing activity from intact human erythrocytes [6,7]. The polar compound was eluted off the C18 reverse phase column with 18% acetonitrile and cross-reacted very weakly with specific antidigoxin antibody. On the other hand, the less polar compound was eluted at 31% acetonitrile and showed a prominent digoxin-like immunoreactivity. In this preliminary communication, we report the different behavior of these two digitalis-like factors in re-

Generation of mice deficient in RNA-binding motif protein 3 (RBM3) and characterization of its role in innate immune responses and cell growth

The activation of innate immune responses is critical to host defense against microbial infections, wherein nucleic acid-sensing pattern recognition receptors recognize DNA or RNA from viruses or bacteria and activate downstream signaling pathways. In a search for new DNA-sensing molecules that regulate innate immune responses, we identified RNA-binding motif protein 3 (RBM3), whose role has been implicated in the regulation of cell growth. In this study, we generated Rbm3-deficient (Rbm3(-/-)) mice to study the role of RBM3 in immune responses and cell growth. Despite evidence for its interaction with immunogenic DNA in a cell, no overt phenotypic abnormalities were found in cells from Rbm3(-/-) mice for the DNA-mediated induction of cytokine genes. Interestingly, however, Rbm3(-/-) mouse embryonic fibroblasts (MEFs) showed poorer proliferation rates as compared to control MEFs. Further cell cycle analysis revealed that Rbm3(-/-) MEFs have markedly increased number of G2-phase cells, suggesting a hitherto unknown role of RBM3 in the G2-phase control. Thus, these mutant mice and cells may provide new tools with which to study the mechanisms underlying the regulation of cell cycle and oncogenesis.

Further evidence for the dissociation of digoxin-like immunoreactivity from Na+, K+-ATPase inhibitory activity

The effects of adrenalectomy or nephrectomy, carried out one hour previously, on the levels of endogenous digitalis-like factors were determined in rat plasma. Factors were assayed by digoxin-like immunoreactivity and direct Na+, K+-ATPase inhibitory activity. Digoxin-like immunoreactivity significantly decreased one hour after bilateral ablation of adrenals, while Na+, K+-ATPase inhibitory activity remained unaltered. There were no changes in either activity one hour after bilateral nephrectomy. These results suggest that digoxin-like immunoreactivity may be derived from the adrenal gland or under adrenal control and the major substances detected by digoxin-like immunoreactivity and direct Na+, K+-ATPase inhibitory activity may be different.